A method for analysis of reducing sugars is known which comprises subjecting a sample containing reducing sugars to liquid chromatography using as a mobile phase an aqueous solution containing boric acid; adding to the eluate, in the flow path, a boric acid aqueous solution containing a basic amino acid such as arginine; and letting it to react with the reducing sugars by heating, then cooling the reaction solution, and measuring its absorbance, or intensity of fluorescence from it under irradiation with excitation light (Patent Document 1). The apparatus used in the method comprises an extended flow path from a column, to which flow path is connected a supply channel for a boric acid aqueous solution containing a basic amino acid, and are attached a heater, a cooling device, an excitation light irradiator, and a device for measuring fluorescence intensity or the like. In this method, the supply channel is indispensable for addition of a boric acid aqueous solution containing a basic amino acid to the eluate from the liquid chromatography.
Further, an improved type of the above method is known, which comprises subjecting a sample containing reducing sugars to liquid chromatography using a mobile phase containing boric acid and a reagent basic amino acid, such as arginine, heating the eluate in the flow path to allow the reagent to react with the reducing sugars, then cooling the reaction solution, and measuring its absorbance, or the intensity of fluorescence under irradiation of the reaction solution with excitation light (Patent Document 2). In this method, as a boric acid aqueous solution containing a basic amino acid is employed as the mobile phase for liquid chromatography, there is no need for providing a supply channel for later addition of a boric acid aqueous solution containing a basic amino acid. These methods for analysis of reducing sugars are called post-column fluorescence detection/boric acid complex anion exchange method.
For detection of reducing sugars eluted from a column, both of the above methods for analysis of reducing sugars utilize production of potent fluorogenic derivatives by heat-reaction of reducing sugars with a basic amino acid, such as arginine in the presence of boric acid (Non-patent Document 1). The fluorogenic derivatives are melanoidins, brown compounds which are produced by a heat-reaction between reducing sugars and a basic amino acid, which is an amino compound (Maillard reaction). They emit light at the wavelength of 430 nm when irradiated with excitation light at the wavelength of 320 nm. For separation of reducing sugars by a column, these methods for analysis of reducing sugars utilize the properties of reducing sugars that they readily bind to boric acid to form an anionic complex ion and this anionic complex ion is retained by an anion exchanger column.
In the post-column fluorescence detection/boric acid complex anion exchange method, an aqueous solution (pH 7-10) is employed, as a mobile phase, which contains a basic amino acid at a concentration of 0.01-5% and boric acid at a concentration of 0.05-0.5 M. Those amino acids which can be employed above are arginine, lysine, histidine, and the like. As any of such basic amino acids, whether it is D-, L-, or DL-form, equivalently produces melanoidins, its enantiomers or racemic mixtures can equally be used in the reaction. Further, it has recently been known to separate sugars using a gradient created in the mobile phase for liquid chromatography based on 0.1 M borate buffer and 0.4 M borate buffer (Patent Documents 3 and 4). Furthermore, a method for separation of sugars utilizing a gradient of an inorganic salt concentration has been developed, in which the concentration of boric acid in the borate buffer used as the mobile phase is set at 50-150 mM and an inorganic salt such as sodium chloride is added to the buffer (Patent Document 5). According to this method, clogging of the piping with precipitating boric acid can be prevented, for the mobile phase contains boric acid only at a low concentration.
In the conventional post-column fluorescence detection/boric acid complex anion exchange method, elution of a sample in liquid chromatography is carried out at temperatures from room temperature to 70° C., and the heat reaction (Maillard reaction) is allowed to proceed at 140-180° C. (Patent Document 2). Since the reaction and elution are performed at such high temperatures and at high boric acid concentrations, such phenomena occur as thermal expansion of the mobile phase and convection in the mobile phase. These phenomena can cause noises in the fluorescence detected by a fluorescence detector in the post-column fluorescence detection/boric acid complex anion exchange method, and can lower the sensitivity of detection. Thus, in the analysis of reducing sugars by such methods, particularly in their quantitative analysis, it has been required to compensate the result of detection by using dedicated software.
Reducing sugars which can be analyzed using the post-column fluorescence detection/boric acid complex anion exchange method are those which undergo the Maillard reaction with basic amino acids, including monosaccharides such as glucose, mannose, galactose, fructose, and rhamnose; oligosaccharides such as maltose and maltotriose; amino sugars such as glucosamine, and galactosamine; and uronic acids such as glucuronic acid.
Examples of analytes of this method include reducing sugars derived from sugar chains of glycoproteins. Reducing sugars as components of sugar chains of glycoproteins include monosaccharides such as mannose, galactose, and fucose; amino sugars such as galactosamine. Such sugar chains sometimes contain mannose-6-phosphate (M6P). M6P in sugar chains is indispensable for a glycoprotein to bind to the mannose-6-phosphate receptor, a membrane receptor.
Some of the enzymes used in the enzyme replacement therapy for lysosomal diseases require M6P in their sugar chains for them to exhibit their pharmacological effect. The reason for this is as follows: those enzymes bind to the mannose-6-phosphate receptor on the cells via M6P in their sugar chains, and then they are taken into endoplasmic reticula in the cells by endocytosis, which then, by fusion with lysosomes, carry them into the lysosomes, and exhibit their pharmacological effect by decomposing their substrate in the lysosomes. Therefore, if they lack M6P, they even cannot be taken in by the cells. Examples of such enzymes include lysosomal acid lipase, acid sphingomyelinase, acid α-glucosidase (acid maltase), and N-acetylgalactosamine4-sulfatase, and these enzymes produced by the recombinant technology are useful in the enzyme replacement therapy for Wolman disease, Niemann-Pick disease, Pompe disease and Maroteaux-Lamy syndrome, respectively.
When it is orally ingested, boric acid, which is used in post-column fluorescence detection/boric acid complex anion exchange method, causes, abdominal pain, cramps, diarrhea, nausea, vomiting, exanthema, and dizziness, and also may cause dermatitis when it is repeatedly or chronically brought into contact with the skin. Further, animal studies have shown that boric acid may have a reproductively toxic effect on humans (Non-patent Document 2). Therefore, the above method poses a substantial impact on the environment due to the resulting waste fluid containing boric acid with such a toxicity.